The mammalian sympathetic nervous system is composed of various cellular tissues having the unique capability of synthesizing postsynaptic neurotransmitter compounds. These compounds are biogenic amines termed catecholamines, a term derived from the catechol, or dihydroxybenzene, nucleus common to each of the compounds. These catecholamines include dopamine, norepinephrine and epinephrine.
Aberrations of the sympathetic nervous system can lead to a wide variety of adverse clinical manifestations. Therefore, accurate and reliable methods for quantifying the concentration of catecholamines in the body are critical to provide adequate monitoring of the system. Further, since these compounds are present in the body in very small amounts, the methods must be highly sensitive, that is, capable of detecting the compounds in very small amounts. The frequency at which these methods are conducted mandate further that they be highly reproducible under laboratory conditions when employing a variety of body tissues and fluids and provide the results quickly in order to facilitate diagnosis.
Radioenzymatic assays are sensitive analytical methods which have found wide use in the quantification of various biogenic amines. These assays are based on the enzymatic methylation of a specified compound to a radiolabeled product by an appropriate enzyme employing radioactive S-adenosylmethionine as the methyl donor. Most of the currently employed radioenzymatic assays lack the sensitivity necessary to quantify catecholamines in important biological samples such as human plasma.
Henry et al. in Life Sciences 16:375 (1975) describe a useful radioenzymatic assay for specifically measuring norepinephrine in tissues, plasma and urine. This method relates to the conversion of norepinephrine to radiolabeled epinephrine employing partially purified bovine adrenal phenylethanolamine N-methyltransferase and tritiated S-adenosylmethionine.
The present invention relates to an improved radioenzymatic assay for norepinephrine comprising the methylation of norepinephrine with purified phenylethanolamine N-methyltransferase to radiolabeled epinephrine employing tritiated S-adenosylmethionine as the methyl donor. The tritiated epinephrine thus obtained may then be conveniently isolated by batch alumina chromatography and any residual unreacted tritiated S-adenosylmethionine may be removed by precipitation upon treatment with phosphotungstic acid. Further, the tritiated epinephrine may be quantified by liquid scintillation spectrometry using a biphasic counting system which employs an ion-pair reagent containing bis(2-ethylhexyl)hydrogen phosphate to extract the tritiated epinephrine into a high efficiency counting environment.